Inhibition of Escherichia coli RNase P by oligonucleotide directed misfolding of RNA

JESSICA L. CHILDS; ALEX W. POOLE; DOUGLAS H. TURNER

doi:10.1261/rna.5780503

Inhibition of Escherichia coli RNase P by oligonucleotide directed misfolding of RNA

¹Departments of Chemistry and
²Pediatrics, and the
³Center for Human Genetics and Molecular Pediatric Disease, University of Rochester, Rochester, New York 14627, USA
⁴Department of Molecular, Cellular, and Developmental Biology, University of Colorado–Boulder, Boulder, Colorado 80309, USA

Abstract

Oligonucleotide directed misfolding of RNA (ODMiR) uses short oligonucleotides to inhibit RNA function by exploiting the ability of RNA to fold into different structures with similar free energies. It is shown that the 2′-O-methyl oligonucleotide, m(CAGCCUACCCGG), can trap Escherichia coli RNase P RNA (M1 RNA) in a nonfunctional structure in a transcription mixture containing RNase P protein (C5 protein). At about 200 nM, the 12-mer thus inhibits 50% of pre-tRNA processing by RNase P. Roughly 10-fold more 12-mer is required to inhibit RNase P containing full-length, renatured RNase P RNA. Diethyl pyrocarbonate modification in the presence of 12-mer reveals increased modification of sites in and interacting with P4, suggesting a structural rearrangement of a large pseudoknot important for catalytic activity. Thus, the ODMiR method can be applied to RNAs even when folding is facilitated by a cognate protein.

Keywords